Fungi antagonistic to xylella fastidiosa

ABSTRACT

The disclosure describes fungal organisms and preparations useful for inhibiting infection by  Xylella  sp. and in the treatment of Pierce&#39;s Disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 61/477,537, filed Apr. 20, 2011, which is incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates generally to methods and compositions useful fortreating plant diseases, in particular infections by pathogens.

BACKGROUND

Xylella fastidiosa (Xf) is a xylem limited bacterium that dwells in awide array of plant species and the foregut of certain xylem feedinginsects. The insects serve to transport Xf from plant to plant whenfeeding. Xf does not harm its host insect and most plant hosts are notharmed by Xf. However certain plant species are susceptible to strainsof Xf and disease can occur.

SUMMARY

The disclosure provides a method for preventing infection and/orprotecting plants from infection by Xylella fastidiosa microorganismscomprising inoculating the plant with a fungal organism selected fromthe group consisting of Cochliobolus sp., Aspergillus sp., Paeosphaeriasp., Ulocladium sp., Dicostroma sp., Geomyces sp., and Cryptococcus sp.,and one baceterial organism Achromobacter sp.

The disclosure provides a method for preventing infection and/orprotecting a plant from infection by Xylella sp. microorganismscomprising inoculating the plant with (i) an anti-Xf endophyticmicroorganism selected from the group consisting of Cochliobolus sp.,Aspergillius sp., Phaeosphaeria sp., Ulocladium sp., Dicostroma sp.,Geomyces sp., Achromobacter sp. and Cryptococcus sp., and anycombination thereof; and/or (ii) an extract comprising an anti-Xf agentthat inhibits Xylella sp. infection. In one embodiment, the Xylella sp.is Xylella fastidiosa. In another embodiment, the anti-Xf endophyticmicroorganism Achromobacter sp. or Geomyces sp. In yet anotherembodiment, the anti-Xf endophytic microorganism comprises a combinationof Achromobacter sp. and Geomyces sp. In yet another embodiment, theplant is a monocotyleyledonous plant. In one embodiment, the plant is adictotyledonous plant. In yet another embodiment, the plant is selectedfrom the group consisting of grape, oleander, oak, almond, peach, pear,citrus, coffee, maple, mulberry, elm, sycamore, and alfalfa. In oneembodiment, the anti-Xf endophytic microorganism is inoculated in thexylem of the plant. In yet another embodiment, the anti-Xf agentcomprises the general structural formula selected from the groupconsisting of: (a) Formula Ia:

wherein,

R² is selected from the group comprising halo, CN, O, OH, NH, NH₂, S,SH, and CH₂;

R³-R⁴ are each individually selected from the group comprising H, halo,hydroxyl, cyano, thiol, amino, optionally substituted (C₁-C₆)alkyl, andoptionally substituted (C₁-C₆)alkenyl; and

R⁶ is selected from the group comprising H, optionally substituted(C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl, and optionallysubstituted heterocycle;

(b) Formula Ib:

wherein,

R² is selected from the group comprising halo, CN, O, OH, NH, NH₂, S,SH, and CH₂;

R³ is individually selected from the group comprising H, halo, hydroxyl,cyano, thiol, amino, optionally substituted (C₁-C₆)alkyl, and optionallysubstituted (C₁-C₆)alkenyl; and

R⁶ is selected from the group comprising H, optionally substituted(C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl, and optionallysubstituted heterocycle;

(c)

In one embodiment, the anti-Xf agent is administered by a syringe intothe xylem.

The disclosure also provides a composition for carrying out the methodsabove, wherein the composition comprises at least one anti-Xf endophyticmicroorganism and/or at least one anti-Xf agent. In another embodiment,the at least one substantially purified anti-Xf endoyphtic microorganismis selected from the group consisting of Cochliobolus sp., Aspergililussp., Phaeosphaeria sp., Ulocladium sp., Dicostroma sp., Geomyces sp.,Achromobacter sp. and Cryptococcus sp. and any combination thereof. Inanother embodiment, the composition is an aqueous media. In oneembodiment, the composition comprises an anti-Xf agent having thegeneral structural formula selected from the group consisting of: (a)Formula Ia:

wherein,

R² is selected from the group comprising halo, CN, O, OH, NH, NH₂, S,SH, and CH₂;

R³-R⁴ are each individually selected from the group comprising H, halo,hydroxyl, cyano, thiol, amino, optionally substituted (C₁-C₆)alkyl, andoptionally substituted (C₁-C₆)alkenyl; and

R⁶ is selected from the group comprising H, optionally substituted(C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl, and optionallysubstituted heterocycle;

(b) Formula Ib:

wherein,

R² is selected from the group comprising halo, CN, O, OH, NH, NH₂, S,SH, and CH₂;

R³ is individually selected from the group comprising H, halo, hydroxyl,cyano, thiol, amino, optionally substituted (C₁-C₆)alkyl, and optionallysubstituted (C₁-C₆)alkenyl; and R⁶ is selected from the group comprisingH, optionally substituted (C₁-C₆)alkyl, and optionally substituted(C₁-C₆)alkenyl, and optionally substituted heterocycle;

(c)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B show inhibition of Xylella fastidiosa with different fungalcultures. A) Both Geomyces sp. and Achromobacter sp. suppressed PDdevelopment in grapevine cuttings cv. ‘Merlot’ mechanically inoculatedwith Xf. Following inoculation, plants were rated weekly on a 0-5disease scale with 0=healthy and 5=dead. B) Geomyces sp., Achromobactersp., and Aureobasidium sp. caused a reduction in Xf titer as compared toplants inoculated with Xf alone.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a plant” includes aplurality of such plants and reference to “the pathogen” includesreference to one or more pathogens known to those skilled in the art,and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

Crop infestations can cause severe damage to crop production and cancause severe economic harm to farmers and consumers. One bacteriumresponsible for plant infections is Xylella, such as Xylella fastidiosa(Xf). Xylella fastidiosa is a gram-negative, xylem-limited bacteriumcapable of affecting economically important crops. The bacterium has alarge host range, including at least 28 families of bothmonocotyleyledonous and dictotyledonous plants. Plant hosts for X.fastidiosa include miscellaneous ornamentals, grape, oleander, oak,almond, peach, pear, citrus, coffee, maple, mulberry, elm, sycamore, andalfalfa, where the bacterium inhabits the plants' xylem. Other strainsof Xylella cause important diseases of peach, citrus, coffee, andnumerous forest tree species. Vectors, such as insects like xylemsap-feeding sharpshooters, acquire the bacterium by feeding on infectedplants and subsequently infect other plants. Xylella can also be grafttransmitted.

Pierce's Disease (PD) is caused by the bacterium Xylella fastidiosa andis spread by certain kinds of sharpshooters known as sharpshooters. Thebacterium resides in the xylem. Insects that feed on xylem sap transmitthe bacteria from diseased to healthy plants and thus are the vector forspread of the disease and bacteria. Plants (e.g., grapevines) showssymptoms of bacterial and infection when the bacteria block the waterconducting system and reduce the flow of water to the leaves. The firstevidence of PD infection usually is a drying or “scorching” of leaves.About mid-growing season, when foliar scorching begins, some or all ofthe fruit clusters may wilt and dry up. The bark on affected canes oftenmatures unevenly, leaving islands of mature (brown) bark surrounded byimmature (green) bark or the reverse. Environmental stress (e.g.,drought) hastens an infected plant's demise.

Pierce's Disease occurs in North America through Central America and hasbeen reported in some parts of northwestern South America and is commonin some California vineyards every year, with the most dramatic lossesoccurring in the Temecula Valley, Coachella Valley, Napa Valley and inparts of the San Joaquin Valley. Economic damages from the disease havebeen estimated to cost as much as $20,000 per acre. During severeepidemics, losses to PD may require major replanting. Currently thereare more than 500 million commercial grapevines in the United States,with 40% of the acreage at risk for significant economic loss. Outbreakof Pierce's Disease in California has also had a major impact on thestate's nursery business due to quarantines imposed in efforts toprevent the spread of the disease.

The manifestation of disease is related to at least two parameters: (1)the successful multi-directional dissemination of Xf within the plantand (2) the plant phenotype that allows a particular strain of Xf toreach high populations within it. These parameters are associated andlargely determine whether a particular strain of Xf will be a harmlessentophyte or a pathogen in any given plant. Plant phenotype and thelevel of affinity each strain of Xf has for a particular plantspecies/strain are the primary factors relating to the disease causingthreshold.

Dissemination of Xf within plants is by three modes: Horizontal movementby secreting pectin degrading enzymes to breaks down the pith membraneof xylem vessel walls allowing Xf to disseminate into adjacent xylemvessels. Xf is also carried along in the xylem flow to disseminateupward, e.g., away from the roots. The final mode of dissemination isfor Xf to crawl against the flow of xylem. Xf uses minute tentacles(phili) to preferably and arduously migrate against the flow of xylem.This mode of dissemination is used by the bacterium to colonize theenduring regions of the plant, e.g., lower branches, trunk, and rootsand thereby help insure the survival of Xf.

Once Xf colonizes the enduring regions of the plant such as the trunk,the infection tends to become chronic. Conversely Xf does not, ofnecessity, create a chronic infection when it infects a plant. If thebacterium does not achieve colonization of enduring regions of the plantand is limited to the green parts such as the canes that are prunedafter harvest then the infection is removed.

Since the mid-1970s, other strains of Xylella fastidiosa have beendiscovered, and almost all of these cause leaf scorching of woodyperennials, such as American elm, maple, mulberry, or plum. In someplants, such as peach and alfalfa, the bacterium slows and stunts plantgrowth. Xylella sp., such as Xylella fastidiosa, are responsible forvariegated chlorosis in citrus, almond leaf scorch disease, phony peachdisease, alfalfa dwarf, and others. Xylella fastidiosa attacks citrusfruits by blocking the xylem, resulting in juiceless fruits of nocommercial value.

Methods of treating or preventing infection by Xylella which have beentried include control of the insect vectors (such as through pesticideand use or physical barriers), destruction of infected plants, andpruning and freezing. Other methods contemplated include the use ofother bacterial species and bacteriophages, for the control of Xylellafastidiosa in host plants, the use of broad-spectrum antibiotics orboosting levels of essential plant bacterial micronutrients such aszinc, iron, copper, and molybdenum that could be toxic to Xylella sp.For example, U.S. Patent Application Publication No. 20050053584,describes the use of a benign strain of Xf (EB92-1) to inhibit avirulent strain of Xf. This form of bio-control can be referred to as“competitive displacement.” The deployment of competitive displacementis problematic, however, because the characteristics that make aparticular strain virulent tend to give the virulent strain competitiveadvantage over the benign strains. Virulence by any one Xf strain is notuniversal to all plants that it infects; rather it is determined by thesuccess of a particular Xf strain in its specific species/strain of hostplant. Thus, when a strain of Xf comes to resides in its preferred planthost any benign strains of Xf that cohabitates the plant are atcompetitive disadvantage. The pre-establishment of the benign strain ofXf in the target plant may offset the benign strains disadvantage. This“head start advantage” tends to weaken over time and eventually thebenign strain will yield to the aggressive strain. Thus, it may berequired to reapply the benign strain after three to five years.

Yet another way to prevent the infection is by genetically modifying thechemistry and structure of the xylem making it uninhabitable for thebacteria (see, e.g., U.S. Pat. No. 6,232,528).

Control of PD with fungi or fungal metabolites is a largely unexploredresearch area, although fungi are known to produce an array of secondarycompounds that have antimicrobial properties (Getha et al., 2009;Mathivanan et al., 2008). Indeed, using fungi as biocontrol agentsagainst plant disease is an active area of research. Some examplesinclude the use of Trichoderma species to control avocado white rootrot, the use of Penicillium oxalicum to control powdery mildew ofstrawberries, and the use of fungal endophytes to control frosty pod ofcacao (Cal et al. 2008; Mejia et al, 2008; Rosa and Herrera, 2009). Inaddition, bio-pesticides that are fungal spore-based are commerciallyavailable.

This disclosure provides microorganisms that inhibit and treat Xfinfection as well as compounds that inhibit, cure and/or treat Xfinfection. The microorganism and compounds were developed through afocused analysis of endophytic fungi and microorganisms in grapevinesand evaluating their potential as biocontrol agents against Xf. Themethods of the disclosure characterized the grapevines that escapedPierce's Disease in natural vineyard settings, and compared themicrobial population to PD-infected grapevines. Through this processfungi and bacterial microorganisms were identified that are unique toPD-escaped vines. Such fungal endophytes and bacteria possess anti-Xfproperties, likely due to the production of certain metabolites. Onceidentified, the ability of these endophytes and their natural productswere assessed for inhibitory activity against Xf in vitro.

Greenhouse tests were performed to determine if (1) the identifiedendophytic fungi have potential use as prophylactic bio-control agentsfor control by inoculating grapevine cuttings with endophytic,Xf-antagonistic fungi; and (2) if injection of fungal natural productshave curative properties in PD-infected grapevines cuttings.

Seven fungi were identified that were strongly inhibitory to Xf invitro, all of which have been identified to genus level based onribosomal DNA sequences-Aspergillus sp., Cochliobolus sp., Cryptococcussp., Discostroma sp., Geomyces sp., Phaeosphaeria sp., and Ulocladiumsp. In addition, a bacterium (Achromobacter sp.) was identified based on16s ribosomal DNA that strongly inhibits Xf in vitro. This bacterium hasa yeast-like growth habit, and was selected for furthercharacterization. It was only later determined to be a bacterium, butbecause of its strong inhibitory effects was further studied.

Fungal candidates that displayed two features: (1) showed inhibitoryeffect of Xf in in vitro assays; and (2) were heavily sporulating inculture were selected for additional studies. Spore formation is animportant criteria in order to re-introduce these fungal endophytes intogrape cuttings by vacuum filtration or other methods. Because of theirsmall size and shape, fungal spores are more likely to infiltrate andcolonize the plant xylem vessels than fungal hyphae. Fungal spores wereharvested in sterile water and the concentration was adjusted to 10⁵ to10⁶ spores/ml.

Using the methods described above and elsewhere herein, anti-Xfendophytic fungi and microorganisms were identified that are useful fortreating plants having an Xf infection as well as preventing spread orinfection by Xf. As used herein the term “anti-Xf endophyticmicroorganisms” refers to Aspergillus sp., Cochliobolus sp.,Cryptococcus sp., Discostroma sp., Ulocladium sp., Phaeosphaeria sp.,Geomyces sp. and Achromobacter sp.

The anti-Xf endophytic microorganisms of the disclosure and anti-Xfagents isolated therefrom are useful in the treatment of target plantsto confer Xf resistance and/or treatment of infection. Suitable plantsinclude, but are not limited to, ornamentals, grape, oleander, oak,almond, peach, pear, citrus, coffee, maple, mulberry, elm, sycamore,alfalfa, peach, and numerous forest tree species susceptible to Xylellasp. infection such as Xf infection. The plant may be at any stage ofgrowth, including seeds, seedlings, or full plants. In addition, asdiscussed herein, any part of the plant may be inoculated; suitableplant parts include seeds, roots, leaves, flowers, stems, trunks, etc.

In one embodiment the disclosure relates to target plants obtained byartificially introducing an anti-Xf endophytic microorganisms intoplants and plant parts not infected with an anti-Xf endophyticmicroorganisms. In the context of this disclosure, the anti-Xfendophytic microorganisms which is artificially introduced into thetarget plant, is a microorganism that confers Xf infection resistance tothe target plant.

In a one embodiment, a pure culture of an anti-Xf endophyticmicroorganism is used to inoculate plants or plant parts. A “pureculture” in this context means a culture devoid of other culturedendophytic fungi. The culture may be of spores, hyphae, mycelia, orother forms of the fungi, with spores being particularly preferred. Ingeneral, spores are used at 1−5×10³⁻⁸ spores per plant with 1−3×10⁴⁻⁶being common and 1−3×10⁵ being typical. As outlined herein, the anti-Xfendophytic microorganisms of the disclosure may be cultured in a varietyof ways, including the use of plates and liquid cultures.

In another embodiment, the plant may be inoculated by vacuuminfiltration. In this embodiment, a stem or cutting is placed in anaqueous media comprising the anti-Xf endophytic microorganisms and avacuum is applied to draw the aqueous media comprising the anti-Xfendophytic microorganisms into the cutting or stem. Once inoculated thecutting or stem is grown using recognized techniques. Typically thisprocess is performed in a nursery.

The spores or other innoculum may be placed on seed coats, particularlyon seeds of anti-Xf endophytic microorganisms-free seeds (eithernaturally occurring or treated to remove any endophytes). It should benoted that the plants, including seeds, may be inoculated withcombinations of anti-Xf endophytic microorganism cultures.

In another embodiment, an extract of an anti-Xf endophytic microorganismcomprising an agent that inhibits Xf growth or infection is used totreat or inhibit a Xylella sp infection (e.g., a Xf infection thatcauses PD). For example, an extract from an anti-Xf endophyticmicroorganism can comprise an agent that inhibits Xf growth andtransmission. In this embodiment, a culture of an anti-Xf endophyticmicroorganism can be disrupted and crude or purified extract may beapplied to the plant. In one embodiment, the extract or purifiedcomposition is administered to the plant at the site of a Xylella sp.growth (e.g., the trunk or stem).

For example, in one embodiment, an extract of an anti-Xf endophyticmicroorganism of the disclosure was prepare and purified to identify anagent that inhibits Xf growth. The agent was identified as havingstructural Formula I:

wherein,

A¹ and A² are each individually O, S, or NH;

R¹ and R² are each individually selected from the group comprising halo,CN, O, OH, NH, NH₂, S, SH, and CH₂;

R³-R⁵ are each individually selected from the group comprising H, halo,hydroxyl, cyano, thiol, amino, optionally substituted (C₁-C₆)alkyl, andoptionally substituted (C₁-C₆)alkenyl; and

R⁶ is selected from the group comprising H, optionally substituted(C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl, and optionallysubstituted heterocycle.

In a further embodiment, the disclosure provides for a compound havingstructural Formula Ia:

wherein,

R² is selected from the group comprising halo, CN, O, OH, NH, NH₂, S,SH, and CH₂;

R³-R⁴ are each individually selected from the group comprising H, halo,hydroxyl, cyano, thiol, amino, optionally substituted (C₁-C₆)alkyl, andoptionally substituted (C₁-C₆)alkenyl; and

R⁶ is selected from the group comprising H, optionally substituted(C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl, and optionallysubstituted heterocycle.

In another embodiment, the disclosure provides for a compound havingstructural Formula Ib:

wherein,

R² is selected from the group comprising halo, CN, O, OH, NH, NH₂, S,SH, and CH₂;

R³ is individually selected from the group comprising H, halo, hydroxyl,cyano, thiol, amino, optionally substituted (C₁-C₆)alkyl, and optionallysubstituted (C₁-C₆)alkenyl; and

R⁶ is selected from the group comprising H, optionally substituted(C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl, and optionallysubstituted heterocycle.

In a specific embodiment, the agent is selected from the groupconsisting of:

As used herein an anti-Xf agent of the disclosure refers to an agenthaving structural Formula I:

wherein,

A¹ and A² are each individually O, S, or NH;

R¹ and R² are each individually selected from the group comprising halo,CN, O, OH, NH, NH₂, S, SH, and CH₂;

R³-R⁵ are each individually selected from the group comprising H, halo,hydroxyl, cyano, thiol, amino, optionally substituted (C₁-C₆)alkyl, andoptionally substituted (C₁-C₆)alkenyl; and

R⁶ is selected from the group comprising H, optionally substituted(C₁-C₆)alkyl, and optionally substituted (C₁-C₆-alkenyl, and optionallysubstituted heterocycle.

In a further embodiment, the disclosure provides for an anti-Xf agenthaving structural Formula Ia:

wherein,

R² is selected from the group comprising halo, CN, O, OH, NH, NH₂, S,SH, and CH₂;

R³-R⁴ are each individually selected from the group comprising H, halo,hydroxyl, cyano, thiol, amino, optionally substituted (C₁-C₆)alkyl, andoptionally substituted (C₁-C₆)alkenyl; and

R⁶ is selected from the group comprising H, optionally substituted(C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl, and optionallysubstituted heterocycle.

As used herein an anti-Xf agent of the disclosure refers to an agenthaving structural Formula Ib:

wherein,

R² is selected from the group comprising halo, CN, O, OH, NH, NH₂, S,SH, and CH₂;

R³ is individually selected from the group comprising H, halo, hydroxyl,cyano, thiol, amino, optionally substituted (C₁-C₆)alkyl, and optionallysubstituted (C₁-C₆)alkenyl; and

R⁶ is selected from the group comprising H, optionally substituted(C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl, and optionallysubstituted heterocycle.

As used herein an anti-Xf agent of the disclosure refers to an agentselected from the group consisting of:

In one embodiment, a combination of anti-Xf agents may be used to treatan Xf infection or prevent an Xf infection.

Methods are provided for protecting a plant from a pathogen (e.g., an Xfpathogen) comprising applying an effective amount of a compositioncomprising an anti-Xf endophytic microorganism of the disclosure and/oran anti-Xf agent/compound of the disclosure. “Effective amount” isintended to mean an amount sufficient to control a virulent pathogen.

The compositions comprising the anti-Xf endophytic microorganism(s)and/or an anti-Xf agent of the disclosure may comprise a surface-activeagent, an inert carrier, a preservative, a humectant, a feedingstimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protective, a buffer, a flow agent orfertilizers, micronutrient donors, or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematicides,molluscicides, acaracides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular targetpathogens. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, e.g., natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders, or fertilizers. Theactive ingredients of the disclosure are normally applied in the form ofcompositions and can be applied to the crop area, plant, or seed to betreated. For example, the compositions of the disclosure may be appliedduring growth, seeding or storage.

The anti-Xf endophytic microorganism(s) and/or an anti-Xf agent of thedisclosure may be applied simultaneously or in succession with othercompounds. Methods of applying a composition of the disclosure include,but are not limited to, foliar application, seed coating, injection andsoil application. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pathogen.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; carboxylate ofa long chain fatty acid; an N-acylsarcosinate; mono or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include, but are not limited to, inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions of the disclosure can be in a suitable form for directapplication or as a concentrate of primary composition that requiresdilution with a suitable quantity of water or other diluant beforeapplication. The concentration of benign fungal organism will varydepending upon the nature of the particular formulation, specifically,whether it is a concentrate or to be used directly.

A compositions of the disclosure can be applied to the environment of aplant pathogen by, for example, spraying, atomizing, dusting,scattering, coating, injecting or pouring, introducing into or on thesoil, introducing into irrigation water, by seed treatment or generalapplication or dusting at the time when the pathogen has begun to appearor before the appearance of pathogens as a protective measure. It isgenerally important to obtain good control of pathogens in the earlystages of plant growth, as this is the time when the plant can be mostseverely damaged. The compositions of the disclosure can convenientlycontain an insecticide if this is thought necessary.

In one embodiment, an anti-Xf agent of the disclosure can be injectedinto the trunk of a plant (e.g., a grape vine). For example, an 18-gaugesyringe needle can be attached to a syringe barrel and used to piercethe base of the vine with the needle. The injection apparatus can besecured to the vine and left in place. This will allow for a continuousdrip of the solution into the xylem stream of the plant—the niche whereXf dwells. This xylem infiltration technique is used by arborists todeliver insecticides, fungicides or plant growth regulators to achievesystemic translocation of the product into the xylem stream. There areseveral commercial products built on this concept, including SilvaShieldInjectable (Bayer AG), Merit Injectable (Tree Tech, Inc.) and ChemJetTree Injector (ChemJet).

Compositions of the disclosure find use in protecting plants, seeds, andplant products in a variety of ways. For example, the compositions canbe used in a method that involves placing an effective amount of thecomposition in the environment of the pathogen by a procedure selectedfrom the group consisting of spraying, dusting, broadcasting, or seedcoating. The methods of the embodiments may be effective against avariety of plant pathogens.

EXAMPLES

One-year-old canes from grapevines varieties Chardonnay and CabernetSauvignon were sampled. Although apparently healthy, these grapevineswere submitted to the constant disease pressure present in RiversideCounty. Canes were pressure-bombed and 100 μl of the sap was plated ongeneral fungal medium, Potato Dextrose Agar (PDA), amended withtetracycline to inhibit bacterial growth. After 2 weeks of growth atroom temperature, the fungi growing were transferred to fresh PDA mediumin order to obtain pure cultures. Fungal DNA was extracted from thesepure cultures with a Qiagen DNA extraction kit. Following this, theribosomal DNA was PCR-amplified (600 base pairs) and sequenced (forwardand reverse). Fungal taxa were identified after comparing the r-DNAsequence to homologous sequences posted in the GenBank database. Taxawere identified from the sap of these vines, and included Aureobasidium,Cladosporium, Cryptococcus, Cochliobolus and Chaetomium.

Samples were also obtained from four varieties in two vineyards in NapaCounty and one vineyard in Riverside. Grapevine varieties includedChardonnay, Merlot, Riesling and Cabernet Sauvignon. One-year-old caneswere collected including the wood spur from blocks that had bothdiseased and PD-escaped grapevines. Samples were pressure-bombed toextract the xylem sap. Following extraction, 100 μl of the xylem sap wasplated on general fungal medium, Potato Dextrose Agar (PDA), amendedwith tetracycline to inhibit bacterial growth. In addition, wood chipswere excised from the one-year-old cane and spur and were also plated onPDA-tetracycline medium. Fungi were cultured and identified as describedabove. The list of endophytic fungi present in escaped and diseasedgrapevines is presented in Table 1. Cladosporium and Aureobasidium werepresent in all grapevine varieties and were also found in the xylem sap.

TABLE 1 Identification and percent recovery of fungal taxa fromPD-escaped and PD-infected grapevines. Results are based on samplingfrom 3 vineyards in Napa and Riverside County, and include 4 grapevinevarieties (Merlot, Cabernet Sauvignon, Chardonnay, Riesling). Fungi wereisolated from xylem sap and one-year-old cane and spur wood. PercentRecovery Escaped Grapevines Diseased Grapevines Fungal Taxa (n = 37) (n= 30) Cladosporium sp. 63 57 Aureobasidium sp. 59 60 Alternaria sp. 1130 Cryptococcus sp. 14 7 Penicillium sp. 3 3 Geomyces sp. 3 7Biscogniauxia sp. 3 3 Nigrospora sp. 3 3 Peyronellae sp. 5 Drechslerasp. 3 Discostroma sp. 3 Cochliobolus sp. 3 Chaetomium sp. 5 Aspergillussp. 3 Phaeosphaeria sp. 3 Pyronema sp. 3 Oidiodendron sp. 3 Diplodia sp.3 Neofusicoccum sp. 3 Epicoccum sp. 7 Phomopsis sp. 3 Fusarium sp. 7Cryptosporiopsis sp. 3 Ulocladium sp. 13 Pezizomycete sp. 7 Didymellasp. 3

Culturable fungal candidates were evaluated in an in vitro inhibitionassay for antagonism against Xf. In brief, Xf liquid cultures wereadjusted to OD₆₀₀ nm=0.1 (approx. 107 CFU/ml). 300 μl of the Xf cellsuspension was added to 3 ml of PD3 medium containing 0.8% agar andbriefly vortexed. This mixture was overlayed onto a petri platecontaining PD3 medium. A #4 size cork borer was flame sterilized andused to cut out a circle of agar containing fungal mycelium from a petriplate containing a fungal culture. This circle was placed onto theplates previously inoculated with Xf. Plates were incubated at 28° C.for 10 days and then observed for an inhibition zone around the fungalcolony. Measurements were taken of the inhibition zone and recorded.Fungal species with inhibition zones were considered inhibitory to Xf.

Crude extracts of the inhibitory fungi were prepared as follows. Agarplugs of 0.5 cm diameter of each fungus were used to inoculate 250 mLliquid media, and the fungi cultivated at room temperature with shaking.After 7 days, each culture was extracted with three portions of 125 mLethyl acetate, the extracts dried over sodium sulfate, and the solventremoved in vacuo.

Xf cultures were prepared as described above. Crude extracts from thedifferent inhibitory fungi were re-suspended in sterile ethyl acetate toa concentration of 2 mg/ml. Volumes corresponding to a total extractmass of 1 mg, 0.1 mg, and 0.01 mg were pipetted onto sterile paper discsand allowed to dry in a laminar flow hood. Once dry, the paper discscontaining the crude extracts were placed onto the Xf cultures andincubated at 28° C. for 7 days. Following this, plates were observed fora halo of inhibition around the paper disc.

Grapes cuttings var. Merlot of 2 buds were vacuum infiltrated with thefungal spore suspension, planted and placed in the greenhouse. Controlplants were infiltrated with sterile water only. Shoots arising from theplanted cuttings were inoculated with X. fastidiosa (Temecula strain) bymechanical needle inoculation. A sub-sample of plants were notinoculated to determine if the concentration of fungal spores used aredetrimental to the grape cuttings. Planted cuttings were evaluated forPD symptoms.

Two exemplary candidate organisms (Achromobacter sp. and Geomyces sp.)decreased PD symptom severity and Xf titer in greenhouse studies.

Initial studies demonstrated that crude ethyl acetate extracts of theculture supernatants of the biocontrol candidate Cochliobolus sp.possess anti-Xf activity. Using nuclear magnetic resonance spectroscopyand mass spectrometry, this inhibitory activity was traced to a singlemetabolite, which we identified as radicinin (a.k.a., stemphylone).Radicinin is known to be produced by several fungi, includingCochliobolus spp. and is also known to possess antimicrobial activity.

1. A method for preventing infection and/or protecting a plant frominfection by Xylella sp. microorganisms comprising inoculating the plantwith (i) an anti-Xf endophytic microorganism selected from the groupconsisting of Cochliobolus sp., Aspergilllus sp., Phaeosphaeria sp.,Ulocladium sp., Dicostroma sp., Geomyces sp. and Cryptococcus sp., andany combination thereof; and/or (ii) an extract comprising an anti-Xfagent that inhibits Xylella sp. infection.
 2. The method of claim 1,wherein the Xylella sp. is Xylella fastidiosa.
 3. The method of claim 1,wherein the anti-Xf endophytic microorganism Achromobacter sp. orGeomyces sp.
 4. The method of claim 1, wherein the anti-Xf endophyticmicroorganism comprises a combination of Achromobacter sp. and Geomycessp.
 5. The method of claim 1, wherein the plant is a monocotyleyledonousplant.
 6. The method of claim 1, wherein the plant is a dictotyledonousplant.
 7. The method of claim 1, wherein the plant is selected from thegroup consisting of grape, oleander, oak, almond, peach, pear, citrus,coffee, maple, mulberry, elm, sycamore, and alfalfa.
 8. The method ofclaim 7, wherein the anti-Xf endophytic microorganism is inoculated inthe xylem of the plant.
 9. The method of claim 1, wherein the anti-Xfagent comprises the general structural formula selected from the groupconsisting of: (a) Formula Ia:

wherein, R² is selected from the group comprising halo, CN, O, OH, NH,NH₂, S, SH, and CH₂; R³-R⁴ are each individually selected from the groupcomprising H, halo, hydroxyl, cyano, thiol, amino, optionallysubstituted (C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl; andR⁶ is selected from the group comprising H, optionally substituted(C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl, and optionallysubstituted heterocycle; (b) Formula Ib:

wherein, R² is selected from the group comprising halo, CN, O, OH, NH,NH₂, S, SH, and CH₂; R³ is individually selected from the groupcomprising H, halo, hydroxyl, cyano, thiol, amino, optionallysubstituted (C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl; andR⁶ is selected from the group comprising H, optionally substituted(C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl, and optionallysubstituted heterocycle;


10. The method of claim 9, wherein the anti-Xf agent is administered bya syringe into the xylem.
 11. A composition for carrying out the methodof claim 1, wherein the composition comprises at least one anti-Xfendophytic microorganism and/or at least one anti-Xf agent.
 12. Thecomposition of claim 11, wherein the at least one substantially purifiedanti-Xf endoyphtic microorganism is selected from the group consistingof Cochliobolus sp., Chaetomium sp., Aureobasidium sp., Ulocladium sp.,Dicostroma sp., Geomyces sp., Aspergillus sp., Phaeosphaeria sp.,Achromobacter sp and Cryptococcus sp. and any combination thereof. 13.The composition of claim 12, wherein the composition is an aqueousmedia.
 14. The composition of claim 11, wherein the compositioncomprises an anti-Xf agent having the general structural formulaselected from the group consisting of: (a) Formula Ia:

wherein, R² is selected from the group comprising halo, CN, O, OH, NH,NH₂, S, SH, and CH₂; R³-R⁴ are each individually selected from the groupcomprising H, halo, hydroxyl, cyano, thiol, amino, optionallysubstituted (C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl; andR⁶ is selected from the group comprising H, optionally substituted(C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl, and optionallysubstituted heterocycle; (b) Formula Ib:

wherein, R² is selected from the group comprising halo, CN, O, OH, NH,NH₂, S, SH, and CH₂; R³ is individually selected from the groupcomprising H, halo, hydroxyl, cyano, thiol, amino, optionallysubstituted (C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl; andR⁶ is selected from the group comprising H, optionally substituted(C₁-C₆)alkyl, and optionally substituted (C₁-C₆)alkenyl, and optionallysubstituted heterocycle; (c)